Sapphire Energy is a biotechnology company developing algae as a scalable low-cost platforms for producing a broad range of proteins with biomedical and biofuels applications. The overall goal of this SBIR application is the development of "enabling technology" capable of producing relatively large amounts of highly purified prokaryotic and eukaryotic integral membrane proteins. Completion of Phase II work would represent validation of a new expression system that uses the chloroplast of the eukaryotic green algae Chlamydomonas reinhardtii as a cost-effective platform for the production of these biologically and pharmaceutically important molecules. Commercialization opportunities include the use of the system for production of important IMP samples for structure-based drug design (SBDD) studies. Production of sufficient amounts of IMPs'remains the most significant bottleneck in efforts to structurally characterize these biologically important molecules, particularly eukaryotic IMPs. The new expression system could also serve as a platform for the industrial scale production of proteins (e.g. microbial membrane proteins) used as vaccines. The immediate goal of Phase I studies is to show that chloroplast thylakoid membranes are suitable substrates for the deposition of recombinant membrane proteins, particularly important mammalian proteins such as transmembrane receptors. Chloroplast thylakoid membranes make up a large portion of algal cells and are quite elastic, capable of harboring large quantities of membrane proteins. Previous studies had generated fusion proteins that contain chloroplast trans-membrane (TM) domains from the photosynthetic proteins, D1 and D2 fused to green fluorescent protein as a reporter. These chimeric recombinant proteins accumulate to relatively high levels in thylakoids, demonstrating that these membranes are capable of harboring engineered recombinant proteins. In this project we will generate recombinant IMPs using a panel of test proteins. We will attempt to understand processes leading to their accumulation in thylakoid membranes with the goal of developing tools, reagents, and protocols for the routine high-throughput over-expression of IMPs. To attain this goal, we propose to achieve three specific objectives;a) Design and Engineer chimeric chloroplast integral membrane fusion proteins, b) Overexpress integral membrane proteins using constructs designed in Aim1, and c) Attempt preliminary purification of a number of these membrane protein that are observed to have high level of expression. Public Health Relevance: The long term goal of this study is the development of a cost-effective platform using the chloroplast of the green algae Chlamydomonas reinhardtii as an expression engine for integral membrane proteins. Successful completion of the project will provide a new and powerful tool to the Structure- Based Drug Design community as it will provide highly purified material for functional and structural studies. Application of the technology will have impact on disease areas such as diabetes, congestive heart failure, cancer, asthma, allergies, high blood pressure and others.